Steatogenesis in adult-onset type II citrullinemia is associated with down-regulation of PPARα

Abstract

SLC25A13 (citrin or aspartate-glutamate carrier 2) is located in the mitochondrial membrane in the liver and its genetic deficiency causes adult-onset type II citrullinemia (CTLN2). CTLN2 is one of the urea cycle disorders characterized by sudden-onset hyperammonemia due to reduced argininosuccinate synthase activity. This disorder is frequently accompanied with hepatosteatosis in the absence of obesity and ethanol consumption. However, the precise mechanism of steatogenesis remains unclear. The expression of genes associated with fatty acid (FA) and triglyceride (TG) metabolism was examined using liver samples obtained from 16 CTLN2 patients and compared with 7 healthy individuals. Although expression of hepatic genes associated with lipogenesis and TG hydrolysis was not changed, the mRNAs encoding enzymes/proteins involved in FA oxidation (carnitine palmitoyl-CoA transferase 1α, medium- and very-long-chain acyl-CoA dehydrogenases, and acyl-CoA oxidase 1), very-low-density lipoprotein secretion (microsomal TG transfer protein), and FA transport (CD36 and FA-binding protein 1), were markedly suppressed in CTLN2 patients. Serum concentrations of ketone bodies were also decreased in these patients, suggesting reduced mitochondrial β-oxidation activity. Consistent with these findings, the expression of peroxisome proliferator-activated receptor α (PPARα), a master regulator of hepatic lipid metabolism, was significantly down-regulated. Hepatic PPARα expression was inversely correlated with severity of steatosis and circulating ammonia and citrulline levels. Additionally, phosphorylation of c-Jun-N-terminal kinase was enhanced in CTLN2 livers, which was likely associated with lower hepatic PPARα. Collectively, down-regulation of PPARα is associated with steatogenesis in CTLN2 patients. These findings provide a novel link between urea cycle disorder, lipid metabolism, and PPARα.

Keywords: JNK; Mitochondrial β-oxidation; NAFLD; PPARα; SLC25A13; Urea cycle disorder.

Fig. 1.

Representative liver histology of CTLN2. Samples were stained with the hematoxylin and eosin (upper and middle rows) or Azan-Mallory method (lower row). Original magnifi- cation;×20 (upper and lower rows) and × 100 (middle row), respectively. Arrows indicate central vein.

Fig. 2.

Down-regulation of hepatic FA β-oxidation enzymes and MTTP in CTLN2. (A) The mRNAs encoding genes associated with FA/TG metabolism. Hepatic mRNA levels were normalized to those of 18S ribosomal RNAand then expressed as fold changes relative to those of the normal group. (B) Immunoblot analysis of ACADM, ACADVL and ACOX1. A representative immunoblot is shown. β-actin was used as the loading control. (C) Band intensities were measured densitometrically, normalized to those of β-actin, and then expressed as fold changes relative to those of normal livers. (D and E) Serum levels of ketone bodies (D) and malondialdehyde (MDA, E). Results were expressed as mean ± SEM. Comparisons between groups were made using the one-way ANOVA with Bonferroni’s correction. Black bar, normal controls (n = 7); Gray bar, mild steatosis CTLN2 group (n = 10); White bar, severe steatosis CTLN2 group (n = 6). *P < 0.05 compared with normal controls; #P < 0.05 compared with mild steatosis group.

Fig. 3.

Down-regulation of hepatic PPARα in CTLN2. (A) Hepatic mRNA levels of PPARs/ RXRA/SREBF1. The same samples used in Fig. 2 were adopted. (B) Immunoblot analysis of PPARs and SREBF1. A representative immunoblot is shown. Histone H1 was used as the loading control. (C) Band intensities were measured densitometrically, normalized to those of histone H1, and then expressed as fold changes relative to those of normal livers. (D) PPARα DNA-binding activity based on an enzyme-linked immunosorbent assay. Binding activity levels were expressed as fold changes relative to those of normal livers. (E) Correlation between PPARA mRNA, PPARα DNA-binding activity, and severity of steatosis. Results were expressed as mean ± SEM. Comparisons between groups were made using the one-way ANOVA with Bonferroni’s correction. Black bar, normal controls (n = 7); Gray bar, mild steatosis CTLN2 group (n = 10); White bar, severe steatosis CTLN2 group (n = 6). ^#P < 0.05 compared with normal controls; ^#P < 0.05 compared with mild steatosis group. Correlation coefficients were calculated using Spearman’s rank correlation analysis.

Fig. 4.

Factors associated with down-regulation of PPARα in CTLN2. (A) Correlation between PPARA mRNA levels and clinical parameters. Correlation coefficients were calculated using Spearman’s rank correlation analysis. (B) Hepatic mRNA levels of genes associated with inflammation. The same samples used in Fig. 2 were adopted. (C) Immunoblot of JNK. The same samples used in Fig. 2 were subjected to immunoblot analysis. (D) Band intensities were measured densitometrically, normalized to those of β-actin, and then expressed as fold changes relative to those of normal livers. Results were expressed as mean ± SEM. Comparisons between groups were made using the one-way ANOVA with Bonferroni’s correction. Black bar, normal control (n = 7); Gray bar, mild steatosis CTLN2 group (n = 10); White bar, severe steatosis CTLN2 group (n = 6). *P < 0.05 compared with normal controls; ^#P < 0.05 compared with mild steatosis group.